BCR-ABL Positive Leukemia Research Articles

Abstract 110: Pro-thrombotic BCR-ABL Tyrosine Kinase Inhibitors Ponatinib And Nilotinib, But Not The Novel Agent Asciminb, Impair Endothelial Cell Wound Healing

Introduction: Tyrosine kinase inhibitors (TKIs) targeting ABL kinase have transformed the treatment of BCR-ABL positive leukemias. First generation imatinib is well tolerated while the newer TKIs, ponatinib and nilotinib, are more effective for leukemia but both substantially increase risk of acute arterial thrombosis, thereby limiting their clinical benefit over imatinib. Asciminib is a new BCR-ABL inhibitor with an unknown safety profile. We previously showed that BCR-ABL inhibitors that increase thrombosis in humans also cause toxicity in endothelial cells (ECs). Hypothesis: We hypothesize that BCR-ABL TKIs that cause EC dysfunction in vitro delay healing at sites of vascular injury in vivo as a potential mechanism of increased risk of thrombosis. Methods: We used the in vitro scratch wound assay and western blot for endothelial nitric oxide synthase (eNOS) phosphorylation in human umbilical vein (HUV)ECs and a mouse carotid artery wire injury model to assess the impact of BCR-ABL TKIs on EC wound healing capacity in vitro and in vivo . Results: ECs were treated with the Cmax concentration in cancer patients, as were mice, as confirmed by serum LC-MS. In vitro , ponatinib and nilotinib significantly decreased HUVEC wound healing compared to both imatinib and asciminib (p<0.001), with no difference between imatinib and asciminib. Ponatinib significantly decreased peNOS compared to both imatinib and nilotinib (<0.05), while asciminib was not significantly different from any of the other TKIs. In vivo , nilotinib significantly decreased carotid EC wound healing compared to imatinib and asciminib (p< 0.05), while ponatinib was not significantly different from any other TKIs. Carotid thrombosis after wire injury was observed in 3/26 mice treated with ponatinib, 5/23 with nilotinib, 1/21 with asciminib, and 0/26 treated with imatinib, with clot-free survival studies ongoing. Conclusions: Ponatinib and nilotinib cause EC toxicity by distinct mechanisms in vitro , which has implications for mitigating their cardiovascular risk. Asciminib causes little vascular toxicity in vitro or in vivo by these assays. Carotid thrombosis developed in some TKI treated mice, suggesting this model can be used to study the link between impaired EC healing and thrombosis.

Concomitant BCR-ABL and JAK2 V617F in a Patient with Myeloproliferative Neoplasm: A Case Report

Myeloproliferative neoplasm (MPN) is a clonal proliferation of the haematopoietic stem cells leading to activated tyrosine kinase signaling activity. Myeloproliferative neoplasm is classified into BCR-ABL positive chronic myeloid leukemia (CML) and BCR-ABL negative MPN which harbors JAK2 V617F mutation in most cases. The genetic combination of BCR-ABL and JAK2 V617F mutation is rare with estimated frequency of 0.4% based on recent study. Herein, we reported a case of a man diagnosed with CML detected by fluorescence in-situ hybdridisation (FISH) showing atypical BCR-ABL fusion pattern in 29% nucleated cells (cut-off levels ≥5% for positive signals) in the presence of JAK2 V617F mutation. However, the BCR-ABL transcript was not detected by the conventional reverse transcriptase-polymerase chain reaction (RT-PCR) method which was specific for major fragments. Interestingly, complete hematological remission was not achieved despite initiation of tyrosine kinase inhibitor (Imatinib). In conclusion, it is imperative to scrutinise CML cases for concomitant JAK2 V617F mutation especially patients with atypical clinical or laboratory findings. Therefore, close monitoring with clinical and ancillary technique especially FISH and molecular methods such as DNA sequencing were crucial to help achieve complete hematological, cytogenetic and deep molecular response alongside targeted therapy.

Combined inhibition of BCR-ABL1 and the proteasome as a potential novel therapeutic approach in BCR-ABL positive acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is a disease of lymphoid progenitor cells with an often aggressive course and is commonly caused by the BCR-ABL fusion gene t(9;22) in adults. This fusion gene encodes a constitutively active tyrosine kinase that can be effectively inhibited by tyrosine kinase inhibitors (TKIs), with imatinib being the paradigmatic agent of this class. However, BCR-ABL+ ALL cells rapidly develop mutations against many of the available TKIs, and consecutive disease relapse still results in an overall unfavorable prognosis for patients with this disease. To date, allogeneic stem cell transplantation is the only known curative therapeutic option for the mostly elderly patients with BCR-ABL+ ALL. The discrepancy between the limited therapeutic armamentarium and the growing therapeutic need in an aging population is therefore a reason to test drug combinations against BCR-ABL+ ALL. In this study, we demonstrate that the combination of TKIs with proteasome inhibitors efficiently and under certain conditions synergistically exerts cytotoxic effects in BCR-ABL+ ALL cells in vitro with respect to the induction of apoptosis. Both sole and combined treatment of BCR-ABL+ ALL with the proteasome inhibitors bortezomib and ixazomib, respectively, and TKI causes a significantly greater reduction in cell viability than TKI treatment alone in both BCR-ABL+ cell lines TOM-1 and BV-173. In BV-173 cells, we observed a significant reduction in cell viability to only 1.26%±0.46% with bortezomib treatment and 1.57±0.7% with combination treatment, whereas cells treated with dasatinib alone still had a viable percentage of 40.58±2.6%. Similar results were obtained when ixazomib was applied to both cell lines, and apoptosis was induced in both cases (93.36%±2.7% apoptotic BV-173 cells when treated with ixazomib and TKI). The combination of TKI and proteasome inhibitor is efficient in vitro, potentially expanding the spectrum of therapeutic options for patients with BCR-ABL+ ALL.

Bcr-Abl drives the formation of Hsp70/Bim PPI to stabilize oncogenic clients and prevent cells from undergoing apoptosis

Although tyrosine kinase inhibitors that inhibit Bcr-Abl kinase activity have shown excellent efficacy in the clinical application of CML patients, it still a challenge to discover alternative targets and novel therapies because of the emergence of Bcr-Abl-independent resistance. Most recently, Hsp70/Bim complex was revealed that driven by Bcr-Abl and testified as a specific target for CML because it folds and stabilizes many Hsp70 oncogenic substrates that mediate CML specific signaling pathways. However, the relationship between Bcr-Abl and Hsp70/Bim complex and how the chaperone complex contributes to Bcr-Abl-driven leukemogenic cells remain unclear. Herein, with the help of S1g-2, a specific small-molecule inhibitor of Hsp70/Bim complex, and Bcr-Abl knockdown to induce a panel of cancer cell lines apoptosis, we illustrated that Bcr-Abl could prevent cells from undergoing apoptosis mainly by driving the formation of Hsp70/Bim complex both in Bcr-Abl positive CML cells and ALL cells. Through cell-based Co-immunoprecipitation experiments, we identified that Bcr-Abl could stabilize oncogenic clients including AKT and eIF4E mainly by driving the formation of Hsp70/Bim complex in Bcr-Abl positive cells. Moreover, we identified that Bim mediates interactions of Hsp70 and Bak in Bcr-Abl positive cells. Together, the target identification of Hsp70/Bim complex could make it as a promising anticancer target for Bcr-Abl positive leukemia treatment.

NGS-Based Screening to Comprehensively Decipher TKIs Resistant Mutations in BCR-ABL1 Positive Leukemias

In BCR-ABL1 positive leukemia, mutations in the BCR-ABL1 kinase domain (KD) are the most common TKIs resistance mechanism. Sanger sequencing (SS) is currently the gold standard for detecting ABL1 KD mutations, but its low detection sensitivity cannot reveal mutations below 20% VAFs (variant allele frequency), and it is challenging to distinguish compound or polyclonal mutations. In recent years, the NGS-based BCR-ABL1 KD mutation screening protocol provides a powerful tool for deciphering complex patterns (compound or polyclonal mutations) and higher detection sensitivity (~2%, hotspot mutation 1%). Based on an inhouse designed NGS-based BCR-ABL1 KD mutation screening protocol, we retrospectively analyzed 348 specimens that undergone SS (ALL, n=164, CML, n=128, CML-BP, n=33, AML, n=3, other, n=20, other includes MAL, B-LBL). The BCR-ABL1 transcripts of 109 cases were 0, 184 cases were >0.1%, and the remaining 55 cases were between 0-0.1%. NGS screening results showed that the average VAF was 32.81% (range, 1.0%-100%), which was not provided by SS. Compared with SS, NGS screening provided a higher mutation-positive rate (22.13% vs. 33.05%, P =0.0013), and Gatekeeper T315I remained the most common BCR-ABL1 KD mutation (12.24% vs. 14.06%, P =0.4513). When the BCR-ABL1 transcript was 0, no mutation was detected in SS; while NGS ABL1-KD discovered mutations in 12 cases (0 vs. 11.01%, P =0.0004) with VAFs lower than 10% except for one S438F (VAF 14.9%) mutation,among them are the well-known F359V and E450G, as well as ten rare mutations, including T277A and E494K. There were also significant differences in mutations between 0 and 0.1% in BCR-ABL1 transcripts (16.36% vs 32.73%, P =0.046). However, when the BCR-ABL1 transcript >0.1%, there was no difference in mutation-positive rate between the two groups (36.96% vs. 46.20%, P=0.072). NGS screening disclosed more multiple mutations (59 vs 82, P =0.03), with significant advantages in detecting >3 mutations (3 vs 18, P = 0.0009). Compound mutations (CMs) were determined in 8 cases (8/384, 2.08%), with the incidence of 24.24% (8/33) in cases which carrying two or more mutations, of which T315I-including CMs were the most common (6/8, 75%). CMs or polyclonal mutation analysis is particularly crucial for severe patients who have received multiple consecutive TKIs treatments, and those with multiple background mutations and a higher risk of progression to clinical resistance. Our results showed that the inhouse designed NGS-based screening protocols could decipher TKIs resistant mutations more comprehensively than SS(Fig 1), and worthy of being implicated in clinical practice. Figure 1 Disclosures No relevant conflicts of interest to declare.

First Results of an Open Label Phase II Study to Evaluate the Efficacy and Safety of Inotuzumab Ozogamicin for Induction Therapy Followed By a Conventional Chemotherapy Based Consolidation and Maintenance Therapy in Patients Aged 56 Years and Older with Acute Lymphoblastic Leukemia (INITIAL-1 trial)

Introduction: Acute lymphoblastic leukemia (ALL) represents around 5% of all newly diagnosed leukemia in patients between 55 and 70 years of age. Despite recent advances especially in younger patients, the prognosis of older patients with ALL remains dismal, even after moderately intensive chemotherapy. Due to increasing toxicity and infection rates in older patients, dose intensification of induction treatment often is no justifiable option. Consequently, new treatment options are needed to improve the survival of older ALL patients. Methods: This open label phase II study of the German Multicenter Study Group on Adult Acute Lymphoblastic Leukemia (GMALL) is currently activated in 14 centers in Germany. Patients aged >55 years with newly diagnosed acute B lymphoblastic leukemia, with the exception of Philadelphia-chromosome or BCR-ABL positive ALL or Burkitt's or mixed phenotype acute leukemia, are eligible. Leukemic blasts must have CD22 surface expression of at least 20%. No previous ALL-specific treatment, with the exception of corticosteroids and/or single dose vincristine and/or up to 3 doses of cyclophosphamide (cycloph.) plus standard prephase treatment are allowed. The 1st induction cycle consists of inotuzumab ozogamicin (InO) 0.8 mg/m2 on day1 and 0.5 mg/m2 on days 8 and 15 together with dexamethasone 10 mg/m2 (days 7-8, days 14-17) and 1 intrathecal injection of methotrexate (MTX), cytarabine (AraC) and dexamethasone (Dexa). The 2nd and 3rd induction cycle consist of InO 0.5 mg/m2 on days 1, 8 and 15 plus intrathecal injection of MTX/AraC/Dexa. Response evaluation is scheduled after each cycle. Patients achieving a complete remission are offered to receive 5 conventional consolidation therapies (3 x ID-MTX/asparaginase; 2 x ID-AraC) and one reinduction therapy (idarubicine/AraC/cycloph./Dexa) in combination with rituximab (for CD20+ ALL), followed by a maintenance therapy with 6-mercaptopurine/MTX. The primary endpoint is is event free survival (EFS) at 12-months follow-up. An event is any of the following: persisting bone marrow blasts after 2 cycles of InO, relapse or death. An event rate of ≤40% at 12 months follow-up is considered to qualify the experimental treatment as very promising for additional testing. Under the assumption of one-sided type I error of 5% and 80% power, 42 evaluable patients were needed for primary endpoint analysis. The INITIAL-1 trial is registered with ClinicalTrials.gov, identifier: NCT03460522. Results: As of July 2020, 31 patients have been included, with induction results available for 29 patients. Median age at initial diagnosis was 64 years (range 56-80 years). Twenty-five patients were diagnosed with a common- and 4 with a pro-B lymphoblastic leukemia. Median CD22 expression on leukemic blasts was 70% (range 21-99%). Due to suspected therapy related liver toxicities, 1 patient received 1 induction cycle and 1 patient 2 induction cycles (both were in remission after the 1st induction). All other patients completed induction therapy and achieved complete remission (CR/CRi) mainly after the 1st induction. Results of minimal residual disease (MRD) measured by PCR are available for 23 patients, with 17 being MRD-negative after induction. So far, 4 events have been reported (2 deaths in remission and 1 relapsed ALL in the 1st year of treatment; one relapsed disease in the 2nd year). With a median follow-up of 242 days, the probability of OS at 1 year is 82.4 %. Two patients received an allogeneic stem cell transplantation in ongoing 1st remission. With regard to adverse events (AEs) during induction therapy 1, 2 and 3, most common AEs ≥CTC 3 reported were leukocytopenia (in 64%, 33% and 13% of all cases, respectively), anemia (54%, 28%, 13%), thrombocytopenia (68%, 17%, 26%) and elevation of liver enzymes (31%, 22%, 20%). Conclusion: Replacement of conventional induction chemotherapy by InO is feasible, results in promising remission rates, and may reduce the risk of early morbidity and lethality, particular in older patients with acute B lymphoblastic leukemia. Disclosures Stelljes: Amgen: Consultancy, Speakers Bureau; Pfizer: Consultancy, Honoraria, Research Funding, Speakers Bureau. Wäsch:Pfizer: Consultancy; Amgen: Consultancy; Janssen: Consultancy. Haenel:Amgen, Novartis, Roche, Celgene, Takeda, Bayer: Honoraria. Lenz:Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy; AstraZeneca: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Agios: Research Funding; AQUINOX: Research Funding; Novartis: Consultancy; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Verastem: Research Funding; Morphosys: Consultancy, Honoraria, Research Funding. Brüggemann:Affimed: Research Funding; Regeneron: Research Funding; Celgene: Consultancy; Roche: Consultancy; Incyte: Consultancy; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Goekbuget:Gilead: Consultancy; Kite: Consultancy; Servier: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Erytech: Consultancy; Amgen: Consultancy, Research Funding. Alakel:Pfizer: Consultancy. OffLabel Disclosure: Inotuzumab ozogamicin for induction therapy (1st line therapy)

Design, synthesis, and biological evaluations of novel 3-amino-4-ethynyl indazole derivatives as Bcr-Abl kinase inhibitors with potent cellular antileukemic activity.

Breakpoint cluster region-Abelson (Bcr-Abl) kinase is a key driver in the pathophysiology of chronic myelogenous leukemia (CML). Broadening the chemical diversity of Bcr-Abl kinase inhibitors with novel chemical entities possessing favorable target potency and cellular efficacy is a current medical demand for CML treatment. In this respect, a new series of ethynyl bearing 3-aminoindazole based Bcr-Abl inhibitors has been designed, synthesized, and biologically evaluated. The target compounds were designed based on introducing the key structural features of ponatinib, alkyne spacer and diarylamide, into the previously reported indazole II to improve its Bcr-Abl inhibitory activity and overcome its poor cellular potency. All target compounds elicited potent activity against Bcr-AblWT with sub-micromolar IC50 values ranging 4.6-667nM. In addition, certain derivatives exhibited promising potency over the clinically imatinib-resistant Bcr-AblT315I. Among the target molecules, compounds 9c, 9h and 10c stood as the most potent derivatives with IC50 values of 15.4nM, 4.6nM, and 25.8nM, respectively, against Bcr-AblWT. Interestingly, 9h showed 2 folds and 3.6 times superior potency to the lead indazole II and 10c, respectively, against Bcr-AblT315I. Molecular docking of 9h pointed out its possibility to be a type II kinase inhibitor. Furthermore, all compounds, except 9b, showed highly potent antiproliferative activity against the Bcr-Abl positive leukemia K562cell (MTT assay) surpassing the modest activity of lead indazole II. Moreover, the most potent members 9h and 10c exerted potent antileukemic activity against NCI leukemia panel, particularly K562cell (SRB assay) with GI50 less than 10nM, being superior to the FDA approved drug imatinib. Further biochemical hERG and cellular toxicity, phosphorylation assay, and NanoBRET target engagement of 9h underscored its merits as a promising candidate for CML therapy.

Effects of Glycyrrhetic Acid on Human Chronic Myelogenous Leukemia Cells.

Chronic myelogenous leukemia (CML) is a type of blood cancer that is initially treated with imatinib (first Abl kinase inhibitor). However, some patients with CML develop imatinib resistance. Several new generation drugs have been developed, but do not overcome this problem. Glycyrrhetic acid (GA) is a plant-derived pentacyclic triterpenoid that exhibits multiple pharmacological properties for the treatment of cancers. The current study aimed to investigate the effects of GA on the K562 cell line (Bcr-Abl positive leukemia). The MTT cell proliferation assay was employed to evaluate the cytotoxic effect of GA compared with imatinib (positive control) against leukemia and normal blood cells. For detection of cell death, an apoptotic/necrotic/healthy assay was performed against the K562 cell line. To investigate the kinase inhibitory activity of GA, the Abl1 kinase profiling assay and a molecular docking study were performed. GA showed Abl kinase inhibitory activity with an IC50 value of 29.2 μM and induced apoptosis in the K562 cell line after 6 h of treatment. The current findings indicate that this class of plant extract could be a potential candidate for treatment of CML.

PARP Inhibition Sensitize BCR-ABL1 Positive Cel

Introduction: BCR-ABL1 play a key role in the development of chronic myelogenous leukemia and a part of Ph1 positive acute lymphoblastic leukemia (ALL). BCR-ABL1 functions as a tyrosine kinase. Whereas, BCR-ABL1 induces genomic instability by downregulation of BRCA1. An innate error of BRCA1, a molecule involved in the homologous recombination repair pathway, causes hereditary breast and ovarian cancer. PARP inhibitor (PARPi) induces synthetic lethality in BRCA defective cell. Therefore, PARP inhibitor is expected to induce efficient cell death with BCR-ABL1 positive cell. In addition, in some previous reports, reduction of PARP1 activity leads to the upregulation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway and BCR-ABL1 tyrosine kinase activates PI3K/AKT pathway. These findings suggest activation of the PI3K/AKT pathway leading to PARPi resistance in BCR-ABL1 positive leukemic cells. Here, we demonstrate that PARP inhibition attenuates BCR-ABL1 mediated leukemogenesis and aberration of factors associated with PARP inhibitor resistance induces cell death to fully transformed leukemic cells. Method: Bone marrow-derived mononuclear cells (MNC) from wild type mice and BCR-ABL1 transgenic (Tg) mice were exposed to PARPi in vivo, and cell death was analyzed Annexin-V positivity. PARPi sensitivity to BCR-ABL1 expressed cell was also investigated in vivo bone marrow transplantation model using mouse hematopoietic stem cell (HCS) infected with BCR-ABL1 expressing retrovirus. To evaluate more precisely the results obtained in vitro and in vivo transplantation model, the genetical approach was also performed. The Parp1 knockout (KO) mice were crossed with BCR-ABL1 Tg mice. Then, Leukemia development and subsequent mouse death were observed. In vitro, HR activity was examined using DR-GFP assay. Genomic instability was investigated using the breakage-fusion-bridge (BFB) generation.Maintenance of HSC as a progenitor of the leukemic cell was analyzed by repopulation activity using colony assay. The growth-inhibitory effect was assessed using BCR-ABL positive cell lines with PARPi and PI3K inhibitor. Results: BCR-ABL1 Tg mice derived MNC showed more hypersensitivity to PARPi. Mouse HCS was infected with BCR-ABL1 expressing retrovirus and transplanted lethally Olaparib or vehicle was administrated intraperitoneal injection one day after transplantation. BCR-ABL1 mediated leukemic death was observed 1 month after transplantation in sham-treated mouse, whereas, Olaparib treated mouse did not develop BCR-ABL1 mediated leukemia. Parp1 KO BCR-ABL1 Tg mice attenuated leukemia development and extended their survival compared with BCR-ABL1 Tg mice. In vitro experiment revealed HR activity was down-regulated by BCR-ABL1 expression in DR-GFP assay. The number of BFB generation was increased in BCR-ABL1 Tg with Parp1 KO background. The colony-forming activity of BCR-ABL1 positive HSC was totally abolished by PARP inhibition after 3 times serial replating, whereas sham-treated HSC retained repopulation activity. However, the effect of PARPi on BCR-ABL positive leukemic cell lines was controversial. Therefore, leukemic cell lines were treated with the PARPi and inhibitors toward the molecules associated with PARPi resistance. As a result, a combination of PARPi with PI3K inhibitor effectively induce cell death in PARPi resistant BCR-ABL1 positive leukemic cell lines. Conclusion and discussion: Tyrosine kinase inhibitor (TKI) is the gold standard of the therapeutic option of BCR-ABL1 positive leukemia. However, TKI monotherapy is not sufficient for complete eradication of leukemic cells. It is highly expected that molecules effectively induce cell death to leukemic cells combined with TKI. PARPi would be one of these candidates. However, PARPi could not induces efficient death in all of the cancer cells that carry the mutation of molecules associated with the HR defect. Comprehensive genetic analysis to reveal PARPi resistance is important for HRR defective cancer cells. Combination therapy of PARPi and inhibitorstoward the molecules associated with PARPi resistance would be a good therapeutic option for Ph1 positive leukemia. Disclosures No relevant conflicts of interest to declare.

BCR-ABL1 p190 in CML: A Minor Breakpoint with a Major Impact

Introduction: The oncoprotein Bcr-Abl has two major isoforms, depending on the breakpoint in BCR gene, p190 and p210. While p210 is the hallmark of chronic myeloid leukemia (CML), p190 occurs in the majority of Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients. p190 occurs as a sole transcript in 1-2% CML patients, associated with distinct features like monocytosis and frequent additional cytogenetic abnormalities (ACA) at diagnosis. It also confers a risk of treatment failure and progression in chronic phase (CP) CML patients. However, the underlying mechanisms are largely unknown. Here we explore the characteristics of p190 and p210 in CML and ALL patients using next generation sequencing, phospho-flowcytometry and high throughput drug testing. Patients and methods: Peripheral blood mononuclear cells (PMNC) were collected at diagnosis from four CP-CML patients harboring p190 isoform from Helsinki University Hospital. Genetic alterations were identified by whole exome sequencing. RNA sequencing was employed to analyze transcriptional profiles of p190 CML (n=3) in contrast to p210 CML patients (n=4). A thorough transcriptional, phosphorylation and drug sensitivity profiling were applied to five p190- and three p210-expressing Ph+ALL patients. Expression alterations were further characterized in two cell line models mimicking BCR-ABL positive leukemia (Ba/F3 and HPCLSK). Phosphorylation profiles were analyzed by flowcytometry and phospho-array (Tyrosine Phosphorylation ProArray, Full Moon Biosystems). For drug sensitivity and resistance testing (DSRT), a custom plate set comprising 75 approved and investigational oncology drugs was used for patient samples and more extensive 528-drugs plates were used for the cell lines. Results: CML patients with p190 had a median age of 72.5 years at the diagnosis (range: 50-80) and all received imatinib as a frontline treatment. Only one patient achieved a fluctuating major molecular response (MMR) by 12 months while the rest of the patients showed primary resistance to treatment and were shifted to a 2nd line TKI, nilotinib (n=2) or proceeded to HSCT (n=1). By exome sequencing we identified 26 variants in p190-CML samples (median per patient=7, range: 2-10), including variants in ASXL1, DNMT3A and KDM4D genes. RNA-sequencing analysis identified 19 and 97 dysregulated genes (Q <0.05) between p190- and p210 in CML and Ph+ ALL cells respectively. In CML, enrichment analysis revealed upregulation of TNF, interferon (IFN), IL1-R and Toll-like receptor (TLR) signaling, TP53-related, cell cycle and apoptosis pathways. Among Ph+ ALL samples, many CML-related genes were upregulated in samples encompassing p210 while IFN-, TP53- and cell cycle-related molecules were upregulated in p190 samples. p190 samples exhibited hyper-phosphorylation of Src kinase compared to p210 samples. DSRT results also revealed increased sensitivity of primary Ph+ ALL-p190 cells to Src-inhibitors (dasatinib and saracatinib), glucocorticoids and MDM2 inhibitors/TP53 activators (SAR405838 and idasanutlin). Regarding cell lines, Ba/F3-p190 showed the upregulation of interferon signaling pathways compared to p210. Src was also hyperphosphorylated in both Ba/F3 and HPCLSK p190 models. In addition to glucocorticoids and Src-inhibitors, compounds blocking the activity of the inhibitors of apoptosis protein (IAP) family were highly effective at reducing the viability of p190 compared to p210 cells in both cell lines. Conclusions: In CML, p190 isoform of BCR-ABL1 is associated with distinct features and should be considered as a high-risk group. Combining clinical, genomic, phosphorylation and drug sensitivity data, we demonstrated that p190 activates specific cancer pathways, notably Src signaling and interferon pathways. Data also suggests that CML patients with p190 could benefit from broad spectrum TKI with Src inhibiting activity or combination of TKI with MDM2- or IAP-inhibitors. Disclosures Heckman: Orion Pharma: Research Funding; Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding. Porkka:Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding. Mustjoki:Novartis: Research Funding; Pfizer: Research Funding; BMS: Honoraria, Research Funding.

Heterogeneous BCR-ABL1 signal patterns identified by fluorescence in situ hybridization are associated with leukemic clonal evolution and poorer prognosis in BCR-ABL1 positive leukemia

BackgroundAlthough extensive use of tyrosine kinase inhibitors has resulted in high and durable response rate and prolonged survival time in patients with BCR-ABL1 positive chronic myeloid leukemia (CML) and acute leukemia, relapse and drug resistance still remain big challenges for clinicians. Monitoring the expression of BCR-ABL1 fusion gene and identifying ABL kinase mutations are effective means to predict disease relapse and resistance. However, the prognostic impact of BCR-ABL1 signal patterns detected by fluorescence in situ hybridization (FISH) remains largely unaddressed.MethodsBCR-ABL1 signal patterns were analyzed using FISH in 243 CML-chronic phase (CML-CP), 17 CML-blast phase (CML-BP) and 52 BCR-ABL1 positive acute lymphoblastic leukemia (ALL) patients.ResultsThe patterns of BCR-ABL1 signals presented complexity and diversity. A total of 12 BCR-ABL1 signals were observed in this cohort, including 1R1G2F, 1R1G1F, 2R1G1F, 1R2G1F, 2R2G1F, 1R2G2F, 1R1G3F, 1G3F, 2G3F, 1G4F, 1R1G4F and 1R4F. Complex BCR-ABL1 signal patterns (≥ two types of signal patterns) were observed in 52.9% (n = 9) of the CML-BP patients, followed by 30.8% (n = 16) of the ALL patients and only 2.1% (n = 5) of the CML-CP patients. More importantly, five clonal evolution patterns related to disease progression and relapse were observed, and patients with complex BCR-ABL1 signal patterns had a poorer overall survival (OS) time compared with those with single patterns (5.0 vs.15.0 months, p = 0.006).ConclusionsOur data showed that complex BCR-ABL1 signal patterns were associated with leukemic clonal evolution and poorer prognosis in BCR-ABL1 positive leukemia. Monitoring BCR-ABL1 signal patterns might be an effective means to provide prognostic guidance and treatment choices for these patients.

A RARE CASE OF DASATINIB-INDUCED CHYLOTHORAX

SESSION TITLE: Tuesday Medical Student/Resident Case Report Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: 10/22/2019 01:00 PM - 02:00 PM INTRODUCTION: Dasatinib is a tyrosine-kinase inhibitor approved for treatment of BCR-ABL positive chronic myelogenous leukemia and Philadelphia chromosome positive acute lymphoblastic leukemia. Dasatinib induced chlyothorax is rare. Here we describe one such case. CASE PRESENTATION: 65-year-old gentleman with chronic myeloid leukemia status post stem cell transplantation currently on tyrosine kinase inhibitor, Dasatinib, was referred to our clinic for further evaluation of left sided pleural effusion. The effusion was noted incidentally on a follow-up echocardiogram to monitor the progression of aortic stenosis. Patient denied shortness of breath, chest plain, dizziness, fevers, or chills. He has been a nonsmoker throughout his life. On examination patient’s vitals were within normal limits. He was saturating well on room air. Physical exam was remarkable for decreased air entry on the left base with stony dull note to percussion. Chest x-ray showed moderate sized left pleural effusion. Routine labs were unremarkable. Patient was suspected to have exudative pleural effusion. Prime differentials were: Non-Hodgkin lymphoma, lung cancer and dasatinib induced pleural effusion. Dasatinib was held. Thoracentesis was performed and 1.5 L of coffee-colored fluid was removed, and sent for analysis. Post-procedure chest x-ray showed improved aeration and small apical left pneumothorax. Follow-up chest x-ray, 3 days later, showed resolution of the pneumothorax with recurrence of left-sided pleural effusion. Patient remained asymptomatic. One week later, patient continued to have bilateral pleural effusions, left more than right, on CT chest. On repeat thoracentesis, 1.5 L of fluid was drained again. Pleural fluid analysis revealed exudative fluid with elevated triglyceride levels of 500 and an elevated cholesterol level of 51, consistent with chylothorax. Patient was started on prednisone 40 mg daily. Dasatinib was stopped and patient’s pleural effusion stabilized on steroids. DISCUSSION: Pleural effusion is a known side effect of dasatinib seen in 5-28% of patients. Dasatinib induced chylothorax is rare with unknown patho-physiology. Proposed mechanisms include inhibition of platelet derived growth factor receptor beta, or block in T lymphocyte function. Management includes withholding dasatinib or in some cases reducing the dose, treating with diuretics and a short course of prednisone. CONCLUSIONS: In patients treated with dasatinib who develop pleural effusion, dasatinib induced chylothorax should be considered as a differential diagnosis. Dasatinib should be discontinued and an alternate tyrosine kinase inhibitor should be considered. Further investigation to define the pathogenesis of dasatinib related chylothorax is warranted given the widespread use of the drug. Reference #1: Z. Baloch et al. 'Dasatinib induced chylothorax in chronic myeloid leukemia.' https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5242121/# Reference #2: Y. Huang et al. 'Dasatinib related chylothorax.'Turkish Journal of Hematology.2015 Mar;32(1):68-72. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4439910/# DISCLOSURES: No relevant relationships by Paramesh Dakshinesh, source=Web Response No relevant relationships by Sana Grover, source=Web Response No relevant relationships by Rajamurugan Meenakshisundaram, source=Web Response

Quinacrine Depletes BCR-ABL and Suppresses Ph-Positive Leukemia Cells

Drug resistance to TKIs and the existance of CML leukemia stem cells is an urgent problem. In this study, we demonstrate that quinacrine (QC) induces apoptosis in BCR-ABL positive CML and acute lymphoblastic leukemia (ALL) cells. Interestingly, QC inhibits the colony formation of primary CD34+ progenitor/stem leukemia cells from CML patients. QC targets RNA polymerase I, which produces ribosomal (r)RNA, involving in protein translation process. Also, QC treatment prolongs CML-like mice survival and inhibits K562 tumor growth in vivo. In conclusion, we demonstrate that QC depletes BCR-ABL protein and suppresses Ph-positive leukemia cells in vitro and in vivo.

Prognostic factors of pediatric patients with Ph-positive acute lymphoblastic leukemia

目的探讨儿童Ph染色体阳性和（或）BCR-ABL融合基因阳性急性淋巴细胞白血病（Ph+ ALL）的临床特征及预后影响因素。方法回顾性分析北京大学人民医院儿科2006年12月至2016年12月收治的68例初治Ph+ ALL患儿的临床资料。采用Kaplan-Meier法进行生存分析，χ2检验及Log-rank检验评估各因素对预后的影响，多因素分析采用Cox回归模型。结果68例Ph+ ALL患儿男女比为2.1∶1，中位年龄8（1~16）岁，中位总生存（OS）时间16.8个月，中位无病生存（DFS）时间13.5个月。早期治疗反应良好率为43.9%（29/66），伴髓系抗原表达组与不伴髓系抗原表达组的早期治疗反应良好率分别为29.6%和61.3%（χ2=5.814, P=0.020）。1个疗程完全缓解（CR）率为86.2%（56/65），早期治疗反应良好组与不良组的1个疗程CR率分别为100.0%和74.2%（χ2=6.680, P=0.003）。诱导化疗期加用伊马替尼组与单纯化疗组的1个疗程CR率分别为94.9%和73.1%（χ2=5.185, P=0.024）。2年和5年的OS率分别为（61.4±7.0）%和（50.8±8.1）%，2年和5年的DFS率分别为（54.6±6.8）%和（48.6±7.3）%。单因素分析显示，Ph+ ALL患儿的2年OS率与初诊WBC、LDH水平、脾脏大小、肝脏大小、是否伴髓系抗原表达、早期治疗反应、1个疗程后流式细胞术检测的MRD水平（或BCR-ABL）、是否应用伊马替尼及不同治疗方案等因素有关（P值均<0.05），2年DFS率与LDH水平、脾脏大小、肝脏大小、是否伴髓系抗原表达、早期治疗反应、1个疗程后流式细胞术检测的MRD水平（或BCR-ABL）、是否应用伊马替尼及不同治疗方案等因素有关（P值均<0.05）。多因素分析显示脾脏肋缘下≥3 cm是影响患者OS（RR=45.7, 95% CI 1.4~1 528.2, P=0.033）及DFS（RR=52.3, 95% CI 1.6~1 725.9, P=0.026）的独立预后危险因素。结论儿童Ph+ ALL具有独特的临床和生物学特征。免疫分型中伴髓系抗原表达患儿的早期治疗反应差。早期治疗反应差的患儿1个疗程CR率低，诱导化疗期加用伊马替尼可明显提高1个疗程CR率。脾脏肋缘下≥3 cm是影响患者预后的独立危险因素。单纯化疗疗效差，化疗联合伊马替尼可以明显提高疗效。

Mutational analysis in BCR-ABL1 positive leukemia by deep sequencing based on nanopore MinION technology

We report a third-generation sequencing assay on nanopore technology (MinION) for detecting BCR-ABL1 KD mutations and compare the results to a Sanger sequencing(SS)-based test in 24 Philadelphia-positive (Ph+) leukemia cases. Our data indicates that MinION is markedly superior to SS in terms of sensitivity, costs and timesaving, and has the added advantage of determining the clonal configuration of multiple mutations. We demonstrate that MinION is suitable for employment in the hematology laboratory for detecting BCR-ABL1 KD mutation in Ph+ leukemias.

CBP/Catenin antagonists: Targeting LSCs' Achilles heel.

Cancer stem cells (CSCs), including leukemia stem cells (LSCs), exhibit self-renewal capacity and differentiation potential and have the capacity to maintain or renew and propagate a tumor/leukemia. The initial isolation of CSCs/LSCs was in adult myelogenous leukemia, although more recently, the existence of CSCs in a wide variety of other cancers has been reported. CSCs, in general, and LSCs, specifically with respect to this review, are responsible for initiation of disease, therapeutic resistance and ultimately disease relapse. One key focus in cancer research over the past decade has been the development of therapies that safely eliminate the LSC/CSC population. One major obstacle to this goal is the identification of key mechanisms that distinguish LSCs from normal endogenous hematopoietic stem cells. An additional daunting feature that has recently come to light with advances in next-generation sequencing and single-cell sequencing is the heterogeneity within leukemias/tumors, with multiple combinations of mutations, gain and loss of function of genes, and so on being capable of driving disease, even within the CSC/LSC population. The focus of this review/perspective is on our work in identifying and validating, in both chronic myelogenous leukemia and acute lymphoblastic leukemia, a safe and efficacious mechanism to target an evolutionarily conserved signaling nexus, which constitutes a common "Achilles heel" for LSCs/CSCs, using small molecule-specific CBP/catenin antagonists.

New Genetic Variation in BCR gene of Major B3a2 Breakpoint BCR-ABL Fusion Gene in Patients with Chronic Myelogenous Leukemia in Yogyakarta, Indonesia

Background:Polymorphic bases in several exons of the BCR gene have been found in several studies of the BCR-ABL fusion gene. Most of the polymorphisms do not have any implications for the primary structure of the BCR-ABL protein. Nucleotide changes are often located in the area close to the fusion region, and therefore may influence primer annealing. Our previous work failed to amplify 15 of 200 samples from BCR-ABL positive chronic myelogenous leukemia (CML) patients using multiplex PCR, the standard method to detect BCR-ABL transcripts used in our institution. The failure was considered due to problems in primer annealing caused by sequence variations. Sequence analysis of BCR-ABL fusion gene breakpoint types in CML patients has never been hitherto performed in Indonesia. Therefore, the aim of this study was to perform sequence analysis of several samples that did not show amplification using the standard method.Methods:Fifteen samples were qualitatively amplified by two-step PCR using inner primers in the 2nd PCR to determine the breakpoint type of the BCR-ABL fusion gene. The 2nd PCR products were used as templates to perform sequence analysis, and the results were compared to those in genbank.Result:Seven and 5 of 15 samples were confirmed as major b3a2 and major b2a2, respectively. One sample featured a combination of b3a2 and b2a2, and 2 samples a combination of b3a2 and b2a2 with an additional fragment at 500bp. Sequence analysis showed 3 sequence variations in the major b3a2 breakpoint. One had been reported earlier (c.3296T>C) but the others (c.3245C>T and c.3359T>C) were novel. Fragments at 500bp were confirmed as b3a2 and similar sequence b3a2 in genbank.Conclusion:This study found two new genetic variations in the BCR gene in BCR-ABL fusion cases.

Use of Tyrosine Kinase Inhibitors Post-Allogeneic Stem Cell Transplant in Patients with Philadelphia or BCR-ABL Positive Acute Lymphoblastic Leukemia: A Systematic Review and Meta-Analysis

Background: The use of tyrosine kinase inhibitors (TKIs) in the treatment of Philadelphia chromosome (Ph+) or BCR-ABL positive acute lymphoblastic leukemia (ALL) has improved remission rates allowing more eligible patients to proceed on to allogeneic hematopoietic stem cell transplantation (alloHSCT). Since many patients relapse following alloHSCT, some guidelines have recommended TKIs post alloHSCT (Couban, 2014, Giebel, 2016). Although some studies have demonstrated the benefits of this strategy, others have indicated that, owing to the immunomodulatory effects of TKIs, this approach may be harmful, leading to higher relapse rates. Given this uncertainty, we conducted a systematic review and meta-analysis of published studies comparing the outcomes of patients post alloHSCT who did or did not receive TKIs.Methods: We searched MEDLINE, EMBASE, Cochrane Database of Systematic Reviews, Cochrane Central Register of Controlled Trials, ACP Journal Club, Scopus and conference abstracts focusing on the period during which TKIs were available (2000 to March 2016). Studies were included if they were retrospective or prospective cohort/randomized studies of adult patients with Ph+ or BCR-ABL+ ALL who underwent alloHSCT and received post-transplant TKIs. Studies that did not include a comparative group of patients who did not receive TKIs, duplicate studies, abstracts later published and studies of only pediatric patients were excluded from further analyses. Two reviewers with ALL expertise independently evaluated all abstracts and performed study selection.Results: The literature search identified 868 unique references, 195 of which were reviewed in detail. Seven studies, 6 retrospective and 1 prospective, comprising 1441 patients met all inclusion criteria (Table 1). Three studies contained one or more pediatric patients and four included patients beyond first complete remission including some with hematological relapse at the time of alloHSCT. All studies had a mix of donor sources including matched related, matched unrelated, mismatched related and mismatch unrelated. Pre-transplant conditioning was myeloablative in 3, reduced intensity in 1 and a combination of the two in the remaining 3 studies.TKIs were administered prophylactically (prior to hematological or minimal residual disease relapse) or pre-emptively (prior to hematological relapse). Data on the TKI used was not available in one study. Amongst the remaining 6 studies, imatinib at 400 - 600mg was the most commonly used TKI with dasatinib being used in 3 studies and nilotinib in 1 study. TKIs were initiated 0.5 - 60 months after alloHSCT and continued for 1-64 months after initiation.Overall, 346 patients received a TKI post alloHSCT while 1095 patients did not. Using a fixed effects model there was no difference in the relapse free survival (HR =0.838, 95% CI 0.678 - 1.036, p=0.102) or overall survival (HR=0.868, 95% CI 0.675 - 1.117, p=0.27) for those who received a TKI compared to those who did not. In contrast, there was a significant increase in relapse rates for those who were treated with TKI post-HSCT (HR 1.204, 95% CI 1.044 - 1.387, p=0.011).Conclusions: While there are compelling theoretical reasons to recommend TKIs post alloHSCT (Couban, 2014), this meta-analysis suggests that their use is not associated with improvements in overall survival or relapse free survival and may be associated with higher relapse rates. However, given the heterogeneous nature of the studies, these results must be viewed with caution. Nonetheless, the results are compelling and highlight the need for larger prospective controlled studies evaluating the role of TKI post alloHSCT in patients with Ph+ ALL. [Display omitted] DisclosuresLiew:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees.

Identification of Bisindolylmaleimide IX as a potential agent to treat drug-resistant BCR-ABL positive leukemia.

Chronic myeloid leukemia (CML) treatment with BCR-ABL inhibitors is often hampered by development of drug resistance. In a screen for novel chemotherapeutic drug candidates with genotoxic activity, we identified a bisindolylmaleimide derivative, IX, as a small molecule compound with therapeutic potential against CML including drug-resistant CML. We show that Bisindolylmaleimide IX inhibits DNA topoisomerase, generates DNA breaks, activates the Atm-p53 and Atm-Chk2 pathways, and induces cell cycle arrest and cell death. Interestingly, Bisindolylmaleimide IX is highly effective in targeting cells positive for BCR-ABL. BCR-ABL positive cells display enhanced DNA damage and increased cell cycle arrest in response to Bisindolylmaleimide IX due to decreased expression of topoisomerases. Cells positive for BCR-ABL or drug-resistant T315I BCR-ABL also display increased cytotoxicity since Bisindolylmaleimide IX inhibits B-Raf and the downstream oncogene addiction pathway. Mouse cancer model experiments showed that Bisindolylmaleimide IX, at doses that show little side effect, was effective in treating leukemia-like disorders induced by BCR-ABL or T315I BCR-ABL, and prolonged the lifespan of these model mice. Thus, Bisindolylmaleimide IX presents a novel drug candidate to treat drug-resistant CML via activating BCR-ABL-dependent genotoxic stress response and inhibiting the oncogene addiction pathway activated by BCR-ABL.

Quantification of BCR-ABL by digital PCR in samples with high copy numbers and rates of molecular responses (as defined by EUTOS) compared to those detected with standard quantitative real-time PCR.

7056Background: The Digital PCR (dPCR) technique has the potential to monitor minimal residual disease in patients with BCR-ABL positive leukemias, since it allows absolute quantification of the ta...